The discovery of the antiviral APOBEC3 (A3) proteins is regarded as one of the most therapeutically promising breakthroughs in HIV/AIDS molecular virology. Four different A3 proteins have the capacity to restrict HIV replication by incorporating into assembling viral particles, physically interfering with the progression of reverse transcription, and deaminating viral cDNA cytosines to uracils. The latter antiviral activity is the defining hallmark of A3-mediated restriction, explaining the genomic strand G- to-A mutations that are frequently observed in patient-derived viral sequences. In the next phase of this ongoing project, we will focus on addressing two related questions, which are regarded as major ?black boxes? in the field. Namely, how are the HIV-1 restrictive A3s regulated post-translationally, and how do these regulatory mechanisms effect the RNA-mediated process of encapsidation, which is an essential step in the overall virus restriction mechanism? These questions will be answered by combining comprehensive proteomics, genetics, and next- generation sequencing datasets to identify specific and shared A3 regulatory factors. A combination of genetics, cell biology, virology, biochemistry, and bioinformatics approaches will be used to delineate underlying molecular mechanisms. These studies are anticipated to lead to novel strategies to therapeutically boost the anti-HIV-1 activities of these enzymes and potentially contribute to a longer-term virus eradication plan. RELEVANCE (See instructions): HIV/AIDS is still a pandemic problem. Targeted innate immune therapies have yet to be developed against this virus. Fundamental research on molecular mechanisms regulating the antiviral APOBEC3 enzymes will stimulate the development of innovative HIV/AIDS therapeutics that work by leveraging this innate immune defense system to extinguish virus replication.